Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470 nm) and methicillin-resistant Staphylococcus aureus
Graphical Abstract
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium, which infects the skin, soft tissues. Infection generally begins as swollen painful red bumps, but can quickly turn into deep, painful abscesses that require surgical draining. Infection can become life threatening when it involves deep tissues, such as bones, joints, surgical wounds, the bloodstream, heart valves and lungs. To combat MRSA infection, a myriad of antibiotics has been used; however, their effectiveness against staphylococci infections has decreased substantially due to the evolution of resistant strains of bacteria [1], [2]. The search for more efficacious remedies and alternative measures to address the problem has intensified. Therapies under investigation include many natural and synthetic products such as antibacterial clay [3], combined honey and antibiotics [4], hyperbaric oxygen [5], photodynamic therapy [6], [7], [8], porphyrine [9] and tetraaryl-porphyrin photosensitizers [10], blue light phototherapy [11], [12], [13], 207 nm UV light therapy [14] and combined blue light and hyperbaric oxygen therapy [15].
Photo-irradiation to inactivate MRSA in vitro is gaining interest. Previously two strains of MRSA are successfully eradicated with 405 nm and 470 nm light in vitro, and more recently, 405 nm, 470 nm and 415 nm blue light have been shown to inactivate cultures of Staphylococcus aureus (both MRSA and methicillin susceptible Staphylococcus aureus (MSSA)), Escherichia coli and other bacterial pathogens [16], [17]; however, the mechanism of bacterial cell death remains unclear. Hence, the focus of this study is to determine blue light induced cellular and intracellular biochemical changes in MRSA. Blue light induced cell death is compared with cell death induced by UV-irradiation, which is known to initiate DNA cleavage. The major finding in this paper indicates that blue light irradiation on MRSA is a complementary or alternative treatment to the existing techniques, and elicits a similar response as UV light, but targets different DNA conformation.
Fourier transform infrared (FTIR) spectroscopy, a nondestructive technique, provides information on quantitative profile of overall biochemical composition of cells and tissues. Recently, the biological and clinical applications of FTIR spectroscopy, driven by advanced computational analysis have been developed [18], [19], [20], [21]. Identification and discrimination of microbial species and subspecies by FTIR spectroscopy has been shown in several studies [22], [23], [24], [25], [26], [27]. The FTIR spectrum of MRSA shows important bands attributed to proteins, lipids and nucleic acids in the biochemical spectral region 1800–900 cm− 1. Other spectroscopic methods including raman spectroscopy [28], [29], [30] and mass spectrometry [31] have also been applied to monitoring and identifying S. aureus species.
Here we present, for the first time, the mechanism of action underlying the blue light inactivation of MRSA by FTIR spectroscopy coupled with principal component analysis followed by linear discriminant analysis (PCA-LDA). Moreover, in this study, the mode of action of UV light and vancomycin against MRSA is investigated. Spectral data were loaded into PCA algorithm to reduce the dimensionality of the data before employing LDA algorithm to reveal clustering [32]. This method enables us to extract important spectral biomarkers differentiating treated and control MRSA. Infrared bands attributed to different conformations of DNA are seen in blue light and UV light irradiated MRSA, when compared to control MRSA. The vancomycin-treated MRSA is the most distinct, and is dominated by the vancomycin absorption bands due to high dosage (40 μg/mL) of the antibiotic to maximally suppress MRSA growth.
Section snippets
Materials and Methods
Five different experimental groups of samples are evaluated using FTIR. Group I: Control I, comprised of untreated MRSA incubated at 37 °C for 24 h (N = 3); Group II: Control II, comprised of untreated MRSA incubated in ambient air for 24 h (N = 3); Group III: Irradiation with 262 J/cm2 of blue 470 nm light, incubated at 37 °C for 24 h (N = 5); Group IV: UV light- irradiated (253.5 nm) MRSA, incubated at 37 °C for 24 h (N = 5); Group V: Vancomycin-treated MRSA, incubated at 37 °C for 24 h (N = 3). The rationale for
Results and Discussion
Colony-forming units (CFU) are determined on control and treated MRSA (collected posterior to FTIR measurements) to characterize the effect of treatment or biological influence on bacteria or colony formation. The results for the two control groups are indicative of functioning MRSA. The light treated groups do not express any bacterial growth while vancomycin-treated bacteria still demonstrates negligible colonies of bacteria as anticipated (Fig. 1). Thus since radiant exposure cumulative
Acknowledgments
Financial support was received from the Stimulus Program to Accelerate Research Clusters (SPARC) of College of Health Sciences, University of Wisconsin-Milwaukee (Grant No. PRJ73CL to J.E and V.V.B), NSF grants CHE-1508240 and CHE-1112433 to C.J.H.
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Joint-first author (V.V.B and E.A contributed equally to this work).